Molecular Mechanisms in Reovirus mRNA Synthesis

呼肠孤病毒 mRNA 合成的分子机制

• 批准号: 6847762
• 负责人: MAX L. NIBERT
• 金额: $ 34.4万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-10 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6847762
• 关键词: RNA binding protein; RNA biosynthesis; Reoviridae; cryoelectron microscopy; genetic transcription; intracellular transport; messenger RNA; posttranscriptional RNA processing; protein structure function; recombinant proteins; virus RNA; virus assembly; virus genetics; virus protein

DESCRIPTION (provided by applicant): The molecular mechanisms by which mammalian orthoreoviruses (reoviruses) and other dsRNA viruses mediate synthesis of their mRNA molecules using virally encoded enzymes packaged within infectious virus particles is a subject of active inquiry because they promise insight into how the steps in mRNA synthesis - plus-strand RNA synthesis (transcription), RNA 5' capping, and RNA transport - occur within the delimited three-dimensional setting of the icosahedral virus particle. A better understanding of these mechanisms in dsRNA viruses should be useful for understanding how related processes are mediated by analogous other viral, microbial, or cellular enzymes and for designing new antiviral, antimicrobial, or anticellular agents directed at them. The long-term objective of our work with reoviruses is to define structure-function relationships for the roles of reovirus proteins in viral replication and effects on host cells and animals. In the current proposal, emphasis is placed on the proteins within the reovirus core (subviral) particle that mediates mRNA synthesis in vitro. Three specific aims are identified, which reflect where further progress appears most promising or necessary to advance understanding in this system. These aims are (1) to determine the structure of the reovirus transcriptase complexes and their arrangement in cores, (2) to define the assembly pathway of the reovirus core shell, and (3) to dissect the functions of the reovirus core proteins in RNA synthesis, capping, and transport. Reovirus particles reconstituted from recombinant proteins expressed using baculovirus vectors play a prominent role in the proposed experiments because of their broad applicability to studies of particle structure, assembly, and functions. Recently determined crystal structures of the transcription- and capping-competent reovirus core particle and the reovirus RNA-dependent RNA polymerase also figure heavily in this proposal by having focused attention on particular areas where more structure information is needed, suggested specific new hypotheses about steps in mRNA synthesis and assembly, and identified specific amino acids in the core proteins to subject to mutagenesis for structure-function testing. The results of these studies will enhance our understanding of the reovirus core as an elegantly designed molecular machine for mRNA synthesis.

描述（由申请人提供）：分子机制 哺乳动物正呼肠孤病毒（呼肠孤病毒）和其他 dsRNA 病毒介导 使用包装在其中的病毒编码酶合成它们的 mRNA 分子 传染性病毒颗粒是积极研究的主题，因为它们承诺 深入了解 mRNA 合成的步骤 - 正链 RNA 合成 （转录）、RNA 5' 加帽和 RNA 运输 - 发生在界定的范围内 二十面体病毒颗粒的三维设置。更好的 了解 dsRNA 病毒的这些机制应该有助于 了解相关过程如何由类似的其他病毒介导， 微生物或细胞酶以及用于设计新的抗病毒、抗菌、 或针对它们的抗细胞药物。我们工作的长期目标 呼肠孤病毒的作用是定义结构-功能关系 病毒复制中的呼肠孤病毒蛋白及其对宿主细胞和动物的影响。 在当前的提案中，重点放在呼肠孤病毒内的蛋白质上 介导体外 mRNA 合成的核心（亚病毒）颗粒。三具体 确定了目标，反映了最有可能取得进一步进展的领域 对于增进对该系统的理解是有希望或必要的。这些目标是 (1) 确定呼肠孤病毒转录酶复合物的结构和 它们在核心中的排列，(2) 定义呼肠孤病毒的组装途径 核壳，以及（3）剖析呼肠孤病毒核心蛋白的功能 RNA 合成、加帽和运输。呼肠孤病毒颗粒重组自 使用杆状病毒载体表达的重组蛋白发挥着重要作用 在拟议的实验中，因为它们广泛适用于研究 粒子结构、组装和功能。最近确定的水晶 具有转录和加帽能力的呼肠孤病毒核心颗粒的结构 呼肠孤病毒 RNA 依赖性 RNA 聚合酶在这一过程中也发挥着重要作用 通过将注意力集中在需要更多结构的特定领域来提出提案 需要信息，提出关于 mRNA 步骤的具体新假设 合成和组装，并鉴定了核心中的特定氨基酸 蛋白质进行突变以进行结构功能测试。结果 这些研究将加深我们对呼肠孤病毒核心作为 设计优雅的用于 mRNA 合成的分子机器。